Plastic bag photo bioreactor
Analysis was performed in a self-constructed plastic bag PBR which operates on the bubble column principle (Fig.1). Briefly, the PBR has a total height of 1.9 m and consists of a metal frame made of aluminum sections (MiniTec, Schönenberg-Kübelberg, Germany). Up to three transparent plastic bags (11 cm diameter) can be mounted on top of the frame. The bags used (CASO Design Braukmann, Arnsberg, Germany) consist of an outer polyamide layer and an inner polyethylene layer that are 15 µm and 135 µm thick, respectively. The bags are closed on the bottom with a foil heat sealer and can be filled with different volumes of water or culture media for algal growth. Six 1.16 m long dimmable light‑emitting diode modules (LEDaquaristik, Hövelhof, Germany) illuminate the algal cells. The bags were aerated with 6 mm tubes made of nylon (RS Components, Frankfurt, Germany). The aeration tubes were inserted from the top of the reaction vessel and then guided to the bottom. The bags were additionally prepared with cord grips (16 mm diameter) that act as ports for sampling or integration of measuring probes, for example. The mixing time experiments were performed at room temperature (23 °C) with 10 L of distilled water and with different gas flow rates (10 L h-1, 50 L h-1, 100 L h-1, 300 L h-1). Mixing was done with nitrogen.
Reference method (pH probe)
Sodium hydroxide was used as a tracer (5 mL of 38 mM NaOH stock solution). The pH probe (EasyFerm Bio HB Arc 120 by Hamilton Bonadaz AG) was immersed 30 cm below the surface of the liquid (upper third of the liquid level) and tracked the changes in pH over time with a measurement interval of 3 sec. A manual syringe with a small tube was used to apply the solution directly to the surface of the fluid. The injection duration was a fraction of second. After the experiment was finished, the bubble column was emptied with a peristaltic pump, rinsed with distilled water, and refilled for the next experiment. Mixing time was noted as the time from addition of the tracer until ±5% of its final stable value (defined as the mean of the last 15 values) was reached. The reference experiments were performed in duplicate.
Optical dye tracing experiments
For these experiments, mixing of a dye was observed with a video camera and subsequently analyzed. The digital camera (Olympus OM-D E-M10 Mark II, Olympus Europa, Hamburg, Germany) was placed in front of the bubble column (mov format, 30 frames/sec). A white background was also placed behind the bubble column to minimize disturbing background signals. The blue food dye (patent blue, E131) (Brauns Heitmann, Warburg, Germany) was added with a manual syringe directly to the surface of the fluid (0.075 g powder was pre-mixed with 5 mL of distilled water). The injection duration was a fraction of second. The bubble columns were emptied after each mixing experiment with a peristaltic pump, rinsed with distilled water, and refilled for the next experiment.
Image analysis based on freeware
The key steps of the image preparation were as follows (Fig.1):
- Extraction and storage of a sequence of images from the original video file (sampled at 30 images/sec) was done automatically at a frequency of one image/sec with the tool VLC media player 3.0.16 (VideoLAN, Paris, France).
- Cropping of the picture was performed automatically with IrfanView 4.58 so that only the bubble columns and a reduced background area are visible.
- Final background removal was performed with the on-line tool removebg (Kaleido AI, Wien, Austria). After automated processing, the files were again stored in the computer’s memory and further analyzed.
- Image analysis and mixing time evaluation rely on quantification of pixel-to-pixel heterogeneity within a given area of interest, which can be performed using the standard deviation (SD) across all pixels used. With this method, the whole image of the bubble column was used after background removal. Standard deviations were determined using Gimp 2.10.24 (GNU Image Manipulation Program). To evaluate the pixel-to-pixel heterogeneity based on the pixel intensities, the GIMP feature “value” was used. This “pseudochannel” simulates a greyscale image. Thus the color information is reduced without an additional work step.